1. Field of the Invention
This invention relates to ligand-receptor binding assay techniques. The determination of the presence or concentration of a ligand analyte that is a member of a specific binding pair (“sbp member”) consisting of ligand and its complementary receptor, in serum or other body fluids relies increasingly upon specific binding assay techniques. These techniques are based on formation of a complex between sbp members in which one or the other of the complex may be labeled with a moiety that produces a signal either directly or indirectly. In the case of competitive specific binding assay techniques, analyte in a sample of fluid being tested for its presence competes with a known quantity of labeled analyte in binding to a limited amount of a complementary sbp member. Thus, the amount of labeled analyte bound to the sbp member varies inversely with the amount of analyte in the sample. In immunometric assays, the analyte is usually a ligand and the assay employs a complementary sbp member and a second labeled receptor, usually an antibody. In such an assay, the amount of labeled receptor associated with the complex is directly related to the amount of analyte substance in the fluid sample. Numerous variations of the above are also used in the detection of analytes such as the use of a receptor for a receptor for the analyte or other binding pairs such as avidin-biotin and the like.
The presence in the sample of one or more interfering substances such as proteins, e.g., albumin, that bind non-specifically to the analyte in question or to a reagent being employed in an assay for such analyte can be a serious factor in compromising the quantitative character of a ligand-receptor assay. The analyte is usually present in very small amounts. An interfering substance may be present in greater amounts and can bind to a significant number of analyte molecules and, thus, reduce assay sensitivity. In many situations, the amount of interfering substance will vary from sample to sample thereby preventing accurate reference to a standard or calibrator normally employed to provide for translating the observed signal into the concentration of the analyte. In order to enhance the accuracy of an assay, it is desirable to diminish or to completely remove the effect of the interfering substance on the observed signal.
Mycophenolic acid (“MPA”) is produced by the fermentation of several penicillium species. It has a broad spectrum of activities, specific mode of action, and is tolerable in large doses with minimal side effects, Epinette, et al., Journal of the American Academy of Dermatololgy 17(6):962-71 (1987). MPA has been shown to have antitumor, antiviral, antipsoriatic, immunosuppressive, anti-inflammatory activities, Lee, et al., Pharmaceutical Research 7(2):161-166 (1990), along with antibacterial and antifungal activities, Nelson, et al., Journal of Medicinal Chemistry 33(2):833-838 (1990). It inhibits inosine monophosphate dehydrogenase, an enzyme in the de novo synthesis of purine nucleotides (Wu, Perspectives in Drug Discovery and Design (1994)2:185-204). Since T and B lymphocytes depend largely upon this de novo synthesis, MPA is able to inhibit lymphocyte proliferation, which is a major factor of the immune response.
The morpholinoethyl ester of MPA, morpholinoethyl (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate (“MPA-M”) is rapidly hydrolyzed in vivo to MPA. Administration of MPA in the form of this ester, greatly improves MPA's bioavailability.
Because MPA is a potent biologically active material, an effective immunoassay could be useful in monitoring its bioavailability. In addition, it may be important to monitor therapeutic drug levels, i.e., optimal drug levels necessary for adequate immunosuppression. Since MPA-M is rapidly hydrolyzed to MPA, an assay for MPA would allow a means of regulating and optimizing MPA-M dosages. It is known that MPA is highly protein-bound in plasma (83>98%) and any factors that alter plasma protein concentrations in patients could affect the accuracy of an MPA assay (Shaw, et al., Therapeutic Drug Monitoring (1995) 17:690-699).
Patients under treatment with MPA and cyclosporin or tacrolimus may be co-administered numerous drugs including, but not limited to, azathioprine, prednisone, methylprednisolone, antivirals, antibiotics, antifungals, cardiovascular agents, diabetic agents and diuretic agents. Many of these drugs have profound effects on metabolism and result in changes in concentrations of various serum/plasma components. There exists, therefore, a potential for interference by these components, either directly or indirectly, in the determination of MPA in the target patient population.
Serum assays in general are limited by the difficulty of variations in plasma protein concentrations in patient populations. Furthermore, variations in sample matrix components that alter free and bound fractions of MPA, such as albumin concentration, can lead to inaccurate immunoassay results without releasing MPA from its bound fraction. In particular, the apparent concentration of MPA would be higher or lower depending on the protein concentration of the sample. For example, in the early post transplant period, albumin concentrations are low relative to a calibrator with normal plasma protein concentrations so that MPA quantitation of these patient samples could be high. A number of factors including time post transplant an metabolic differences due to co-administered drugs or disease states of the patient can result in abnormal protein concentrations. These abnormal protein concentrations may alter the free-to-bound ratio of MPA and, therefore, affect the accuracy of the immunoassay results. Variable recovery as a function of protein concentration of the samples prevents selection of one average protein concentration for calibrators that represents all samples.
Salicylate is known to increase MPA free fraction in normal human plasma when present at concentrations that may be observed in chronic administration of aspirin (Nowak, infra). However, we have found that the use of salicylate as a releasing agent can result in a 25-50% decrease in the total dose-response curve in an MPA enzyme immunoassay. It is also known that 8-anilino-1-naphthalenesulfonic acid (ANS) functions as a releasing agent in immunoassays (Nerli, et al., Arch Int Physiol Biochim Biophys (1994) 102(1):5-8 and Seth, et al., Clin Chem (1975) 21(10):1406-1413. However, ANS has disadvantages because of its background absorbance at 340 nm and its susceptibility to light degradation. The background absorbance is particularly disadvantageous in enzyme immunoassays. However, ANS has been used in certain enzyme assays under conditions where its disadvantages can be tolerated.
The present invention avoids the deficiencies of the above known compounds used as releasing agents in assays for ligands.
2. Description of the Related Art
Nowak, et al., Clin. Chem. (1995)41(7): 1011-1017 discusses mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics.
Langman, et al., Therapeutic Drug Monitoring (1994) 16:802-807 discusses blood distribution of mycophenolic acid.
European Patent 0 218 309 B1 discloses a method for measuring free ligands in biological fluids. Sodium salicylate and 2,4-dinitrophenol were employed to prevent labeled analogs of triiodothyronine and tetraiodothyronine from binding to albumin and thyroid binding pre-albumin.
European Patent Application 0 392 332 A2 discloses a fluorescent polarization Immunoassay and reagents therefor. Various compounds were disclosed for converting a marijuana metabolite, which was bound to serum albumin and other proteins in urine, to free form. These compounds included, among others, ANS, salicylic acid and 5-methoxysalicylic acid.